| Research backgroundSmall intestine is highly sensitive to radiation.High-dose ionizing radiation inhibits intestinal epithelial proliferation,and apoptosis and degenerative necrosis of irradiated cells may occur.As a result,intestinal epithelial integrity is damaged,exposing the intestinal epithelial cells to greater contact with gut pathogens,thereby causing severe intestinal inflammatory response.An in-depth study of radiation-induced intestinal injury following exposure to high-dose irradiation will shed new light on the prevention of enteral radiation disease and the protection for abdominal radiotherapy in clinical practice.Pyroptosis is a form of programmed cell death mediated by caspases.Upon receiving the danger signals,the intracellular receptors form inflammasomes,recruiting and cleaving the pro-Caspase-1.Consequently,the activated Caspase-1 is released for downstream execution,cleaving the pyroptosis effector protein.The pyroptosis effector protein contains one N-terminal domain and one C-terminal domain.The C-terminus of the protein can inhibit the pore-forming activity of the N-terminus,and the N-terminal domain of gasdermin D(GSDMD)can form pores in the cell membrane.Once the hinge area of the full-length protein is cleaved by the activated caspase,the self-inhibition activity of the N-and C-termini is triggered.Therefore,oligomerization of the N-terminal domain occurs,leading to pyroptosis.The pyroptotic cells undergo a series of morphological changes,including cell membrane permeability change,release of cellular contents,and massive entry of extracellular water into the cells via the cell membrane.Finally,the cell lysis and death are induced,triggering the inflammatory response.The chemotherapeutic agents used in the clinic may cause severe side effects by activating the apoptotic pathway of the tumor cells,which restricts the application of the chemotherapeutic agents.For a long time,apoptosis has been considered a mechanism by which the cytotoxic drugs kill the cancer cells.Recent studies have confirmed that pyroptosis can also inhibit tumor cell proliferation.Both apoptosis and pyroptosis are caspase-dependent programmed cell death pathways.Caspase-3 is a common key protein in the apoptosis and pyroptosis pathways.The expression level of the activated tumor suppressor gene GSDME determines which pathway is activated to induce the death of tumor cells.Cells with a high GSDME level undergo pyroptosis in the presence of external stimuli,and those with a low GSDME level undergo apoptosis.GSDME can be located not only downstream of caspase-3 but also upstream,connecting endogenous and exogenous apoptosis and promoting the activation of caspase-3,thereby magnifying the inflammatory response.GSDME-mediated pyroptosis is closely associated with the side effects of chemotherapy and anti-tumor immunity.Radiation can also induce the death of a large number of normal cells.Apoptosis,autophagy,and necrosis of mouse intestinal cells are observed after large-dose ionizing radiation.The mice die within a short time,and the use of apoptosis,autophagy and necrosis inhibitors do not offer an adequate protection.Several mechanisms of pyroptosis have been found associated with the regulation of radiation-induced intestinal injury.So far,little has been known regarding the molecular mechanism by which the inflammasome-induced pyroptosis is involved in the radiation-induced intestinal injury.Further studies are needed to clarify the relationship between the pyroptosis of the intestinal cells and other cell death pathways,such as apoptosis and autophagy.ObjectivesThere are still a lack of effective treatments against the death of intestinal cells and the injury of intestinal tissues induced by radiation.It has been found that ionizing radiation can cause significant activation of GSDME and cell pyroptosis.The studies on the complex gene regulatory network based on GSDME expression are still limited.A growing body of evidence has shown that ce RNA competitively binds to mi RNA,regulating the expression of mi RNA or m RNA.ce RNA plays a crucial role in cell development and diseases.The present study was mainly concerned with the changes in the physiological functions of intestinal cells induced by ionizing radiation.The relationship between GSDME and pyroptosis was discussed based on the ce RNA network.The protective effect of pyroptosis against the intestinal epithelial injury induced by ionizing radiation and the underlying molecular mechanism were discussed.Methods1.Human intestinal epithelial cells were cultured in vitro.After irradiation at different doses,the pyroptosis indicators were detected.The molecular mechanism by which pyroptosis was involved in the radiation-induced injury of intestinal epithelial cells was studied;2.mi RNAs related to the pyroptosis effector protein GSDME were predicted in mi RDB,Target Scan,mi RWalk,and micro RNA.org.The predicted mi RNAs with high reliability were chosen.Those with a regulatory effect on GSDME were confirmed by RT-PCR and reporter gene assay.A systematic study was conducted on the mi RNA-regulated GSDME expression in radiation-induced intestinal injury;3.ce RNA network related to radiation-induced pyroptosis was constructed.Long non-coding RNA(lnc RNAs)related to the predicted mi RNAs were identified by literature search.It was then determined whether a dose-effect relationship existed between these lnc RNAs and ionizing radiation.Changes in the expression of the GSDME gene were detected after the interference of the identified lnc RNAs.4.The morphology and function of the irradiated cells were studied after inducing GSDME overexpression or inhibiting the associated lnc RNAs;5.The working mechanism of ce RNAs in the radiation-induced pyroptosis was investigated.Results1.Ionizing radiation induced the pyroptosis of intestinal epithelial cells;2.The GSDME protein played a key role in the radiation-induced intestinal pyroptosis;3.In intestinal cells subjected to ionizing radiation,mi R-448 specifically targeted the GSDME gene,inhibiting its expression;4.The NEAT1-miR-448-GSDME axis played an essential role in the radiation-induced pyroptosis of intestinal cells.ConclusionIt was found that ionizing radiation induced the pyroptosis of intestinal epithelial cells,which was mediated by the GSDME protein.mi R-448 specifically targeted GSDME m RNA,inhibiting its expression by binding to the 3’UTR.lnc RNA NEAT1 constituted the ce RNA regulatory axis by competitively binding to mi R-448,thereby regulating GSDME expression. |
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